Physical characterization and - NSF/SRC ERC HomepageERC TeleSeminar 04/22/2010 UTD. Overview ......
Transcript of Physical characterization and - NSF/SRC ERC HomepageERC TeleSeminar 04/22/2010 UTD. Overview ......
Physical characterization and in vitro cytotoxicity screening
of single-walled carbon nanotubes (SWNTs)
Ruhung Wang, Ph.D.
Bionano Science Group
University of Texas at Dallas
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Overview
I. Introduction
II. Physical Characterizations of SWNT Dispersions
III. In vitro Cytotoxicity Assays
IV. Removal of Toxic Components
V. Summary
VI. Works in Progress
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What are single–walled carbon nanotubes (SWNTs)?
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Javey, A. , ACS Nano (2008) 2: 1329-1335
SWNT
Roll-Up
n
m
Graphene Sheet
Variety of SWNT tube types
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www.carbonwall.com
Methods used for SWNT synthesis
• Natural, incidental, and controlled flame –Nanotubes produced by burning methane, benzene.
• Laser ablation –Blast a composite of graphite and metal catalyst with a laser
• Arc discharge –Nanotubes produced on graphite electrodes during an arc discharge
• Chemical vapor deposition (CVD) –Nanotubes grow on metal catalyst particles as carbon-containing gas break down in a heated reactor. CoMoCAT CVD HiPco CVD Super-growth CVD
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Not all SWNT products are created equal.
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Most commercially available SWNT products are mixtures
SWNTs (metallic & semi-conducting)
Metal catalysts / catalyst support
Non-tubular carbon (NTC) species
By-products of purification / modifications
Representative properties of SWNT products
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SWNT product
CG100 SG65 SG76 HiPco-R AP P2 P3*
Synthesis
methodCoMoCAT-CVD
HiPco -
CVDArc discharge
ManufactureSouthwest NanoTechnologies
(Sigma Aldrich)Unidym Carbon Solution Inc. (CSI)
Carbon (%) > 90 ~ 65 40 – 60 80-90 80-90
Metal(%) < 10 (Co, Mo) <35 (Fe)~ 30
(Ni, Y)
4-7
(Ni, Y)
5-8
(Ni, Y)
SWNT (%) ≥ 65≥ 77
>50% (6,5)
≥ 77
>50% (7, 6)NA NA
Raman D/G <0.06 NA NA
Length (nm) 450-2300 450-2000 300-2300 100-1000[1000-
5000]
[500–
1500][500-1500]
Diameter
(nm)0.7-1.3 0.7-0.9 0.7-1.1 0.8-1.2 [2-10] [4-5] [4-5]
In-house characterizations of purchased SWNT products
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UTD in-house SWNT analysis: TGA – SWNT and metal contents
AFM – individual nanotube length
and diameter
Raman – nanotube identification
and quality assessment
ICP-MS – identify metal catalysts
and quantification
UV-Vis-NIR – identify nanotubes
varieties and quality
FTIR – identify nanotube surface
impurities/modifications
No mandatory SWNT product specification
Product specification data provided may not be adequate and/or accurate.
High quality control is crucial for applications in high-tech industries.
No ecological information available
I. Introduction • > 120 varieties of SWNT tube types
• Commercial SWNTs are synthesized by AD or CVD methods
• SWNT products are mixtures of
{SWNTs, Metals, NTC, others …}
• Vendor provided data are incomplete/inaccurate
• No ESH data available
• In-house characterization of purchased SWNT products is essential.
The SWNTs produced by different manufacturers are unique.
Know your SWNT product before you use it!
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I. Introduction
II. Physical Characterizations of SWNT Dispersions• Dispersion protocol
• AFM analysis
• Concentration
• Vis-NIR Absorbance Spectroscopy
• Raman analysis
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• SWNT material –
10 mg purchased SWNT product
• Solutions –
10 mL HB (10 mM HEPES, 10 mg/mL BSA, pH 7.4)
• Sonication –
Bath sonication (40K Hz, 120W, cooling coils)
4 hours in 4OC cold room (bath temperature: 3-12oC)
Up to 8 samples, 10 mL each, prepared per batch
1 mg/mL SWNT suspensions
• Centrifugation –
20,000 g, 5 min, collect top 90% supernatant
20,000 g, 30 min, collect top 95% supernatant
SWNT dispersions, TBD mg/mL
• Storage –
4OC, stable for longer than one month
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SWNT Dispersion protocol
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AFM analysis of SWNT Dispersion
0
10
20
30
40
Perc
ent
of
SWN
Ts (
%)
Categories by Length (nm)
CG100 Dispersion Length Analysis
n=195
The average SWNT length: 244 nm
• Samples: a) SWNT suspension (1 mg/mL) b) SWNT dispersion (conc. TBD)
• Lanes 2-6:
Load 100, 200, 300, 400, and 500 ng of SWNT suspension to construct a
pixel intensity vs. SWNT concentration calibration curve
• Lanes 7-9:
Load 5,10,15 µl of SWNT dispersion
• Apply electrophoresis –
SWNTs are too big to enter the gel , form a dark band at liquid-gel interface
• Scan the gel and quantify the dark band pixel intensity –
Band darkness <≈> pixel intensity <≈> SWNT /NTC amount
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Quantify SWNT/NTC concentration in dispersions
Lane 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
Before electrophoresis After electrophoresis
Wang et al., Anal Chem. (2009) 81 (8): 2944-52.
SWNTs
Dye
Physical characterizations of SWNT dispersions
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SWNT
DispersionCG100 SG65 SG76 HiPco-R AP P2 P3*
Synthesis
methodCoMoCAT HiPco Arc discharge
Conc.
(ug/mL)230 10 301 63 258 31 401 55 241 29 289 41 786 61
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Vis-NIR absorbance analysis of SWNT dispersions
C
B
A
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Raman analysis of SWNT dispersions
0
5000
10000
15000
20000
25000
30000
35000
1200 1300 1400 1500 1600 1700
Inte
nsi
ty (C
CD
Co
un
t)
Raman Shift (1/cm)
CG100
SG65
SG76
HiPco
AP
P2
P3
|--- D-Band ---|
0
1000
2000
3000
4000
100 150 200 250 300
RBM|---- G-Band ----|
I. Introduction
II. Physical Characterizations of SWNT Dispersions• Hydrophobic SWNTs can be individually dispersed in
biological aqueous solution
• Concentrations of SWNT/NTC in dispersions can be quantified using SDS-PAGE system
• Quality, quantity, and the tube types of SWNTs in dispersions can be measured and analyzed by optical absorption and Raman Scattering.
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I. Introduction
II. Physical Characterizations of SWNT Dispersions
III. In vitro Cytotoxicity Assays• Toxicity assessment
• IC50
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• Cell culture – Normal Rat Kidney (NRK) cell line
• SWNT dispersions – 100 µg/mL in culture medium
• Incubation – 4,000 cells per well
4 wells per sample per assay
37oC, 3 days
• Assays –
Cell CountsCount cell number in each well using a Coulter counter
Crystal Violet StainingStain cells with Crystal Violet dye which binds DNA
Elute bound dye with 10% acetic acid
Transfer the elusion to a 96-well plate
Measure CV dye intensity at A590 using a micro-plate reader
(A590 <≈> bound CV dye <≈> DNA amount <≈> cell number)
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In vitro SWNT cytotoxicity assay protocol
In vitro cytotoxicity assessment of SWNT dispersions
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0
25
50
75
100
% o
f C
on
tro
l
Toxicity of P3-SWNT dispersion as a function of time
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0
25
50
75
100
0 1 2 3
Ave
rage
Ce
ll C
ou
nts
pe
r W
ell
(% o
f C
on
tro
l)
Incubation Time (Day)
Control
CG100
P2
P3
Toxicity of P3-SWNT Dispersion as a function of concentration
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P3-SWNT dispersion IC50 = 76.5 4.9 µg/mL
0
50
100
1 10 100
Ce
ll P
rolif
era
tio
n (
% o
f C
on
tro
l)
P3-SWNT Dispersion (µg/mL)
Expt-1
Expt-2
Expt-3
IC50 : the concentration of a drug that is required for 50% inhibition in vitro.
I. Introduction
II. Physical Characterizations of SWNT Dispersions
III. In vitro Cytotoxicity Assays• 2 methods were used for toxicity assessment
• 7 purchased SWNT products were tested
• 1 SWNT product is toxic: carboxylated P3-SWNT product
• P3-SWNT toxicity is time dependent
• P3-SWNT toxicity is concentration dependent:
IC50 = 76.5 µg/mL
Only the carboxylated SWNT product contains toxic material.
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I. Introduction
II. Physical Characterizations of SWNT Dispersions
III. In vitro Cytotoxicity Assays
IV. Removal of toxic components• Remove toxic material by filtration
• Analysis of filtration materials
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Separation of the Toxic Components from P3 Carboxylated SWNTs Product
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Step 1:
Filter 5 mL diluted P3 dispersion (in HEPES+BSA solution) through a 0.22 µm membrane and collect the Filtrate.
Step 2:
Pass 5 mL HEPES+BSA solution through the same membrane and collect the solution passed through.
Step 3:
Extract material retained on the membrane with 2 mL HEPES+BSA solution, termed Recovery.
HEPES+BSA Solution
WashFiltrate
P3 c-SWNTs Dispersion
Recovery
HEPES+BSA Solution
Vis-NIR absorption analysis of P3 filtration materials
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0
0.5
1
1.5
2
400 500 600 700 800 900 1000 1100
Ab
sorb
ance
Wavelength (nm)
Diluted P3
Recovery
Filtrate
Wash
HEPES+BSA
AFM and Raman Analysis of P3 Filtration Materials
Starting c-SWNTs Filtrate Recovered Material
0
2,500
5,000
7,500
10,000
12,500
15,000
1000 1200 1400 1600 1800
Inte
nsi
tyWavenumber (cm-1)
0
2,500
5,000
7,500
10,000
12,500
15,000
1000 1200 1400 1600 1800
Inte
nsi
ty
Wavenumber (cm-1)
0
2,500
5,000
7,500
10,000
12,500
15,000
1000 1200 1400 1600 1800
Inte
nsi
ty
Wavenumber (cm-1)
D/G = 0.57 D/G = 0.80 D/G = 0.17
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Filtration Purified Carboxylated SWNTs are Not Cytotoxic
0
25
50
75
100
Control Staring Material (P3 c-SWNTs)
Filtrate Recovered Material
Ce
ll P
rolif
era
tio
n
(% o
f C
on
tro
l)
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I. Introduction
II. Physical Characterizations of SWNT Dispersions
III. In vitro Cytotoxicity Assays
IV. Removal of toxic components
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• The commercially purchased carboxylated P3 SWNT product appear to contain amorphous carbon fragments.
• The toxic components in P3 SWNT product, smaller than 0.22 µm in size, can be removed by filtration.
• The recovered materials, larger than 0.22 µm in size,
are not toxic.
V. Summary
Commercially obtained SWNT products require physical and chemical characterization to ensure quality, purity, and lack of toxicity.
7 purchased SWNT products were tested for toxicity in a model mammalian cell culture system using 2 different assays.
Only one SWNT product tested was toxic as a function of exposure time and concentration.
The toxic component in the P3 SWNT product could be removed from the SWNTs by filtration.
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I. Introduction
II. Physical Characterizations of SWNT Dispersions
III. In vitro Cytotoxicity Assays
IV. Removal of toxic components
V. Summary
VI. Works in progress
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Related research projects
1. SWNT carboxylation <=?=> toxicity • carboxylation process creates more amorphous carbon fragments
• majority of amorphous carbon fragments are removed by filtration
Are COOH-SWNT products from different sources toxic?
Does the extent of carboxylation correlate with toxicity?
2. Expand study to other carbon based nano materials • double-walled carbon nanotubes (DWNTs)
• multi-walled carbon nanotubes (MWNTs)
• graphene oxide
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Worsley et al., J Am Chem. Soc. (2009) 131 (508): 18153-8.
Acknowledgements
UTD BioNano Science Group PIs: Dr. Rocky Draper Dr. Inga H. Musselman
Dr. Paul Pantano Dr. Gregg R. Dieckmann
Dr. Steven O. Nielsen
Co-authors : Dr. Carole A. Mikoryak Lab members: Vasanth M. Siruvallur
David Bushdiecker Prashant Raghavendran
Austin Swafford
Synyoung Li BioNano students
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BioNano Group